[35S]GTPγS Binding as an Index of Total G-Protein and Gα-Subtype-Specific Activation by GPCRs

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through -promoting guanine nucleotide exchange on Gα subunits of heterotrimeric G proteins to release Gα-GTP and free βγ-subunits. In membrane preparations, GTP can be substituted by ³⁵S-labeled guanosine- 5'-O-(3-thio)triphosphate ([³⁵S]GTPγS) and on agonist stimulation a quasi-stable [³⁵S]GTPγS-Gα -complex forms and accumulates. Separation of [³⁵S]GTPγS-Gα complexes from free [³⁵S]GTPγS allows differences between basal and agonist-stimulated rates of [³⁵S]GTPγS-Gα complex formation- to be used to obtain pharmacological information on receptor-G-protein information transfer. Further, by releasing Gα-subunits into solution following the [³⁵S]GTPγS binding step, Gα-subunit-specific antibodies can be used to investigate the Gα-protein subpopulations activated by receptors by immunoprecipitation of [³⁵S]GTPγS-Gα complexes and quantification by scintillation counting. Here, we describe a total [³⁵S]GTPγS binding assay and a modification of this method that incorporates a Gα-specific immunoprecipitation step.